Production and use of endophytes as novel inoculants for promoting enhanced plant vigor, health, growth, yield  reducing environmental stress and for reducing dependency on chemical pesticides for pest control

ABSTRACT

A process and method for the production of endophytes as plant inoculant products, specifically  Clonostachys rosea  strain 88-710, for the promotion of plant vigor, health, growth and yield are disclosed. The endophyte,  Clonostachys rosea  strain 88-710 produces a fungal conidial preparation by utilizing a discrete solid substrate fermentation system, namely Potato Dextrose Agar or Malt Extract Agar. Additionally, the endophyte,  Clonostachys rosea  strain 88-710, can act as an inoculant to stimulate and have an additive effect with  rhizobium  bacteria on the production of nitrogen fixing nodules on legumes and growth enhancement e.g. beans, soybeans, peas and alfalfa. As well,  Clonostachys rosea  strain 88-710, can combine with rooting hormones, e.g. indole-3-butyric acid (IBA) to provide inoculant and rooting benefits to cuttings/transplants of plants.

FIELD OF INVENTION

The present invention relates to a production and use process wherebyendophytes are identified as inoculant products, specifically the fungusClonostachys rosea strain 88-710, provides for:

-   (1) Unique inoculant feature/benefits for the promotion of plant    vigor, health, growth and yield.-   (2) A process for making the product(s) that provides for various    levels of highly viable/stable spores and mycelia material in stable    formulations that prevents the degradation of a living endophyte C.    rosea.-   (3) The product formulation allows/enables practical use and    application of the product(s) to seeds, roots, stems, leaves,    flowers, bulbs, etc of plants as a water based sprayable    formulations or as a dusts for other uses e.g. seed treatment and    insect vectoring.-   (4) Allows the product (as an endophyte) to be formulated into    value-added products that provide unique feature/benefits to provide    natural induced plant resistance to fungal diseases such as powdery    mildew, botrytis, root rots, seed seed decay, wilt diseases and or    insects/mites.-   (5) and allows the product (endophyte) when applied to roots, stems,    leaves, flowers, wounds or cut surfaces of plants to act as an    inoculant ‘enhanced site occupier’ within the tissues of plants.-   (6) and endophyte product provides improved root, leaf, stem and or    vegetative bud (flowers) growth to plants as well as seed    germination and-   (7) for reduction of environmental or cultural stress by providing    induced natural plant resistance to plants e.g. root stresses due to    trimming, pruning, cutting, wounding or other stresses with the net    result of:-   (8) Improved crop quality and faster development to marketability of    the crop.-   (9) A strain of Clonostachys rosea i.e. 88-710 with a unique genetic    coding that differentiates the endophyte reproductively from other    fungal strains of Clonostachyus rosea or Gliocladium roseum.-   (10) An endophyte Clonostachys rosea strain 88-710 acts as an    inoculant to stimulate and have an additive effect with soil    rhizobium bacteria for the production of nitrogen fixing nodules on    legumes for growth enhancement e.g. soybeans.

DEFINITIONS

The term endophyte as described in this invention is defined as (1) a“site occupier” i.e. endophytes “or fungi or bacteria that formsymptom-less infections, for part or all of their life cycle withinhealthy leaves and stems of plants” (Definition by Hawksworth et al,1995). (2) a microbe living symbiotically or commensally in a hostplant.

The term inoculant as described in this invention is defined in severalFederal, or State regulations as (1) “substances other than fertilizers,manufactured, sold or represented for use in the improvement of thephysical condition of the soil or to aid plant growth or crop yields”(Canada Fertilizers Act) or (2) “a formulation containing pure orpredetermined mixtures of living bacteria, fungi or virus particles forthe treatment of seed, seedlings or other plant propagation material forthe purpose of enhancing the growth capabilities or disease resistanceor otherwise altering the properties of the eventual plants or crop” (Adhoc European Working Group, 1997) or (3) Soil or plant inoculants shallinclude any carrier or culture of a specific micro-organism or mixtureof micro-organisms represented to improve the soil or the growth,quality, or yield of plants, and shall also include any seed orfertilizer represented to be inoculated with such a culture (New YorkState 10-A Consolidated Law) or (4) “meaning any chemical or biologicalsubstance of mixture of substances or device distributed in this stateto be applied to soil, plants or seeds for soil corrective purposes; orwhich is intended to improve germination, growth, quality, yield,product quality, reproduction, flavor, or other desirablecharacteristics of plants or which is intended to produce any chemical,biochemical, biological or physical change in soil (Section 14513 of theCalifornia Food and Agriculture Code).

The term mycoparasite as described in this invention is defined as (1) Afungus that parasitizes anther fungus (2) a fungal parasitism of anotherfungus or fungi (3) an organism which attacks and feeds on fungi.

The term hyperparasite as described in this invention is defined as anorganism that is parasitic on other parasites.

Clonostachys rosea strain 88-710 defined as the specific endophytefungus isolated and screened from field samples in Ontario and producedunder the novel production technique described in this invention.

The term EndoFine® is defined as the product formulation/brand name forproduct formulations of Clonostachyus rosea strain 88-710 and is aregistered trademark of Adjuvants Plus Inc.

ADJ 702 is defined as the product code for a specific formulation ofClonostachys rosea strain 88-710 containing natural components in theform of calcium, carbonate, potassium salts, and natural emulsifiers(food grade proteins, lactose) that provides rapid wound healing andsite occupation benefits as an inoculant that in turn, also providesadditional plant inoculant protective benefits against a wide range ofplant diseases, insects and mites e.g. powdery mildew, two-spottedmites, aphids.

The term Bionectria ochroleuca is referred to as the referenceMycologia: Vol. 91, No. 2, pp. 365-385.1999. Classification of themycoparasite Gliocladium roseum in Clonostachys as C. rosea, itsrelationship to Bionectria ochroleuca, and notes on otherGliocladium-like fungi. Hans-Josef Schroers, Gary J. Samuels, Keith A.Seifert, and Walter Gams

BACKGROUND OF THE INVENTION

Endophytes such as the fungus Clonostachys rosea formerly calledGliocladium roseum, have been most commonly described in the literatureas a “site occupier” i.e. endophytes “or fungi or bacteria that formsymptom less infections, for part or all of their life cycle withinhealthy leaves and stems of plants” (Definition by Hawksworth et al,1995).

Sample data on endophytes such as the fungus Clonostachys rosea orGliocladium roseum (as it was formerly known), indicate that the fungusoccurs worldwide in a variety of soils e.g. Europe, North America, LatinAmerica, etc. However not all strains of Clonostachys rosea are the samein morphology, reproductively or have the same genetic material. Strain88-710 described in this invention is unique reproductively to thefungus species Clonostachyus.

The background for the classification of Clonostachys rosea is describedin the reference i.e. (Mycologia: Vol. 91, No. 2, pp. 365-385.1999.Hans-Josef Schroers, Gary J. Samuels, Keith A. Seifert, and Walter Gams.

Because the common soil fungus and mycoparasite Gliocladium roseumdiffers from the type species of Gliocladium, G. penicillioides, inmorphology, ecology, teleomorph, and DNA sequence data, it is classifiedin a separate genus, Clonostachys. Penicillium roseum is the oldestavailable name for G. roseum and is recombined as C. rosea. Penicilliumroseum, described from potato in Germany, is neotypified by a conidialisolate originating from a fungal substratum in European soil. Bychoosing this strain as neotype for P. roseum the epithet is formallylinked to the common soil fungus used in the biocontrol of fungal plantpathogens. The anamorph of Bionectria ochroleuca (Hypocreales) ismorphologically indistinguishable from C. rosea; both morphs areredescribed. Bionectria is generically distinct from Nectria s. s. andis the appropriate genus for species of the Nectria ochroleuca group.The anamorph genus Gliocladium s. s. is associated with teleomorphs inSphaerostilbella and Hypocrea series Pallidae. With the separation ofClonostachys from Gliocladium and Bionectria from Nectria the genericclassification reflects natural relationships. A generic circumscriptionis proposed for Clonostachys and compared with Gliocladium. Nectriopsissporangiicola and Roumegueriella rufula are related to Bionectria buthave distinct Gliocladium-like anamorphs. Based on morphologicalfeatures, Rhopalocladium myxophilum gen. et sp. nov. is proposed for theanamorph of N. sporangiicola. The anamorph of Roumegueriella rufula isgenerally found in association with the teleomorph and is referred to asGliocladium-like.

Not all endophytes respond to plants in the same way. Endophytes areknown to infect healthy plants e.g. via wounds e.g. mechanical damagecaused by insects or diseases and in some instances research hasdocumented certain endophytes to provide some measure of plant diseaseor control of insect pests.

However, little or no documentation has been provided that showendophytes to act as inoculants to promote plant health, growth or vigorand capable of providing “natural inoculant induced plant resistance(IIR)” to environmental stresses. Moreover, little or no information hasbeen provided to show that specific endophytes such as the strain 88-710of Clonostachys rosea when acting as an inoculant can also co-exist withother beneficial fungi, bacteria or viruses to provide additive orstimulated growth benefits.

For example, there is no known data or information that demonstratesthat a fungus esp. an endophyte e.g. Clonostachys rosea strain 88-710can act as an inoculant to stimulate the production of nitrogen fixingnodules on legumes e.g. soybeans. Until this invention, it was thoughtthat only naturally occurring bacteria e.g. Rhizobium were capable ofproducing and stimulating the production of nitrogen fixing nodules onlegume plants and reducing dependency on chemical fertilizers. Thisinvention teaches that Clonostachyus rosea strain 88-710 can act as aninoculant to stimulate and be additive (beneficially interactive withnitrogen fixing bacteria) in the production of nitrogen fixing noduleson legumes e.g. soybeans

This invention describes new examples/traits about endophytes such asthe strain 88-710 of Clonostachys rosea that are novel, inventive,useful that teaches something new. The fungus called Clonostachys rosea(C. rosea) although it occurs in virtually all parts of the world, invarious strains/forms and in various climate zones, it has most oftenbeen characterized as a closely (morphological) counterpart ofPenicillium with slimy/sticky conidia. The role and function ofPenicillium spp. as inoculants, is well documented but the inoculantfunction and mode of action of its closely related relative i.e. theendophyte C. rosea has not been documented, quantified or demonstrateduntil this invention.

Until recently, the role or mode of action of endophytes such as C.rosea was not fully understood and it was thought that all strains of C.rosea exhibited only biocontrol modes of action; most such studies wereconducted in the presence of plant diseases and not in the absence ornear absence of plant diseases. The data appeared to only measure orindicate that C. rosea or Glioclaidium roseum (as it was previouslyknown) may control various fungal infections of plants as a biocontrolagent e.g. mycoparasite or hyperparasite by exuding antibiotics,metabolites or mycoparasitic enzymes.

However, as this invention shows, endophytes such as C. rosea, strain88-710 do not directly control fungal or bacterial diseases nor do theyhave a direct mitigating role of controlling plant pests i.e. diseasesor insects, mites, etc. by exuding antibiotics, toxic metabolites orenzymes. Rather as the novelty of this invention will demonstrate, isthat C. rosea acts as a true inoculant on plant tissues to promote planthealth, size, root growth or increases yield and also can aid inreducing or eliminating the use of chemical fungicides while as itimparts natural induced plant resistance with suitable formulations toenvironmental stresses such as plant diseases, moisture reduction,insects and mites. It does this mainly by having the ability to rapidlycolonise plant tissue (living or senescing tissue), imparting resistanceto the plant while occupying the tissue thus denying such tissue toinfection by disease organisms, and other stresses while assisting theplant to uptake nutrients.

The current invention also describes C. rosea strain 88-710 as providingplant health benefits to plants irrespective of whether a pathogen ispresent, hence true non-biocontrol features.

The mode of action of C. rosea strain 88-710 thus appears instead to beone of it ability to provide a rapid first presence as an “inoculantsite occupier” to leaves, stems, and roots thereby promoting plantvigor, plant health, growth and stress reduction such as the preventionof root biomass loss during stem trimming. This ability to help plantsretain good root mass/growth helps plants ward off or re-cover from theshock of trimming (bonsai effect) resulting in faster re-generation ofstem growth and better utilization of nutrients. It also acts in thesame way to overcome other stresses to plants whether those stresses areenvironmental or cultural.

Moreover, endophytes such as C. rosea, strain 88-710 are thought to workby enhancing the solubility/availability of plant nutrients e.g.phosphorus (Reference: Tilak, K. V. B. R et al “Diversity of PlantGrowth and Soil Health Supporting Bacteria”, July 2005 Current ScienceVol 89 N01). This novel invention will show that the strain 88-710 ofClonostachyus rosea acts to enhance nitrogen, phosphorus, and potassiumuptake as well micronutrients in plants and can actcomplementary/additive to nitrogen fixing Rhizobium bacteria. Moreover,this invention demonstrates that endophytes such as C. rosea can becombined with other beneficial bacteria, or fungi due to the fact thatthe 88-710 strain of C. rosea is not toxic to such organisms i.e. doesnot exude toxic metabolites, antibiotics or enzymes as to otherbio-control fungi or mycoparasites. One of the key feature benefits ofinoculants is that true inoculants can be ad-mixed together for mutualbenefit, unlike bio-control agents.

This invention will show that some endophytes such as C. rosea strain88-710, are more specialized in that they possess the ability topenetrate inside root tissues, leaves, stems, etc of plants and havedirect access to organic compounds present in the apoplast”. Once insidethe apoplast of plant tissues, endophytes such as C. rosea can surviveand exude volatile exudates/compounds that promote plant health as wellinduce the plant to protect itself from various stress factors via IIR(inoculant induced resistance) e.g. as disease infections (botrytis,fusarium, Pythium, etc.) and or insect/mite attack. C. rosea has theunique inoculant feature/benefit of living within living and orsenescent plant tissue and either remaining dormant or using that tissueas food to provide plants with nutrition even though the plant tissuemay have been wounded by a disease, mite or insect. All of these arehitherto new, inventive, novel and useful features for endophytes actingas inoculants.

As indicated, Endophytes may also promote growth directly by thefixation of nitrogen alone or in combination with nitrogen fixingbacteria and or solubilization of minerals such as phosphorus, andprovide the production of non-toxic siderophores that solubilize orsequester iron and other key micronutrients (manganese, zinc, etc.). Theabove inventive, novelty, application and new teachings of thisinvention show the endophyte inoculant benefits of promoting plantvigor, health and growth. In order for endophytes e.g. C. rosea, strain88-710 to exhibit such benefits, the endophyte e.g. C. rosea needs to bepresent in sufficient quality/quantity and in a useful bio-availableemulsifiable form as an effective and rapid ‘site occupier’ i.e. stable,robust spores in a deliverable formulation. The claims of this inventionprovide those rapid “site occupier” characteristics.

This invention also describes a novel, inventive and use application(improved sporulation and stability), for the production of an endophytee.g. C. rosea that optimizes manufacture, concentration and thereby theperformance of an endophyte product, for various uses as aninoculant(s).

Moreover naturally occurring endophytes such as C. rosea, strain 88-710(which was originally identified from some 1400 different naturallyoccurring field isolates (samples) from Ontario strawberry fields, wouldhave remained a relatively ineffective obscure endophyte/inoculant wasrendered effective by this invention to provide the above inoculantplant health benefits and induced plant disease/insect/mite reductionfeature/benefits by being available in a practical, useful and effective(as a site occupier) product formulations against major plantpathogens/insect pests e.g. Botrytis cinerea (grey mould). Without thesekey production and physical traits, endophytes such as C. rosea mightremain relatively ineffective as inoculants.

This invention is also particularly applicable where wounding duringpruning, cuttings, wounding, root trimming, transplanting/transplantsand the like occurs.

The use of endophytes as inoculants, such as C. rosea, has the potentialto offer a number of feature/benefits to help counter inadequacies,periodic failures and concerns associated with present pest control andcostly agronomic cultural practices. These include cultural andsanitation measures, regulation of the microclimate, and reduce theheavy dependency on synthetic fungicides/insecticides with consequencessuch as fungicidal/insecticidal resistance, environmental and humanexposure/loading and the general reduction of production costs, thereduction of energy needs/costs (hence reduction of greenhousegases/contaminants), time to market, and enhancement of crop quality.

A patent has been granted e.g. U.S. Pat. No. 6,495,133 Dec. 17, 2002using a certain strain coded ATCC # 74447 of Gliocladium roseum as abiocontrol agent for controlling diseases caused by fungal pathogense.g. Fusarium, Ascochyta, Pythium, Rhizoctonia in plants for treatmentof seeds, soil or plants (pea, bean, canola, wheat, barley,horticultural and ornamental plants). Patents have also been grantede.g. U.S. Pat. No. 6,475,566 Nov. 5, 2002 or U.S. Pat. No. 5,344,252Jul. 9, 1996 for the protection of lumber against sapstains or fungi.However, none of these patents identified the novel usefulness of thisinvention for Clonostachys rosea, strain 88-710 or endophytes asbeneficial inoculants for promoting plant vigor, health and growth inplants in the absence of disease or insect pressures or this combinedwith induced plant resistance to environmental stresses by plantsthemselves.

In particular, outlined in this invention, the need to identify andprovide the correct physical properties to an endophyte for a high levelof inoculant “rapid site occupation” to ensure performance in plantsthat in turn provides for improved leaf, stem and root growth andthereby reducing stress factors (environmental as well as cultural) ofplants.

There have also been examples of other patents granted for endophytese.g. U.S. Pat. No. 6,815,591 Nov. 9, 2004, or U.S. Pat. No. 5,723,720Mar. 3, 1998, that provide plants via seed with imported insectresistance and drought tolerant traits into plants. However, again thesepatents do not document inventiveness, novelty, or usefulness regardingenhanced plant growth, overall plant health enhancement or these traitscombined with plant inoculant resistance to stresses e.g. diseases orthe ability to enhance endophyte product formulations to provide addedvalue inoculant feature/benefits to allow plants themselves to resistattack from pests.

Other patents e.g. U.S. Pat. No. 4,550,527 Nov. 5, 1985 describe methodsusing special soil mixtures and containers of how to best infect rootsof plants with beneficial mycorrhizal fungi for the purposes ofimproving plant health but these methods are not related to endophytesor to specific endophytes such as C. rosea esp. strain 88-710 or tospecific feature/benefits of site inoculation and growth enhancement.

The present invention overcomes drawbacks in the prior art and teachessomething new for information in the public domain. The drawbacks areovercome by a combination of the features of the main claims. Thesub-claims disclose further advantageous embodiments of the inventionand may also overcome drawbacks in the prior art. The present inventionprovides for many of the key feature/benefits the marketplace is seekinge.g.:

-   -   (1) Natural Endophyte products such as C. rosea, strain 88-710        that can act as plant inoculants that provide unique        feature/benefits for the promotion of plant vigor, health,        growth and yield including stimulation of legume plants to        produce nodules for fixation of nitrogen (until this invention        an unknown mode of action for fungal endophytes)    -   (2) Natural crop enhancement products that can increase yields,        quality and reduce growing periods amidst environmental and        cultural stresses e.g. drought, better utilization of nutrients,        thus lowering energy costs and consequences of inefficient use        of energy.    -   (3) Little or no risk of increased environmental or contaminate        hazards due to the use of conventional agrochemicals/pesticides        or fertilizers.    -   (4) Effective production of a natural bio agent in a stable        useful formulation(s) that imparts/enhances unique physical        ‘site inoculant occupation’ within plant tissues that can be        organically certified for a growing market need i.e. OMRI        certification, that in turn    -   (5) Imparts growth and plant protectant properties as a plant        inoculant resistance effect against diseases, wounds or in a        combined action for improved crop production that is unlikely to        develop into “lack of performance resistance” as do chemicals        pesticides.    -   (6) Provide natural wound healing benefits as an inoculant that        can in turn in suitable formulations (that contain natural        salts/emulsifiers) can provide plant protective benefits against        a wide range of plant diseases, insects and mites e.g. powdery        mildew, two-spotted mites, aphids.

There is strong market interest to have as agronomic tools bio agentsthat are naturally occurring for use in improving crop production andquality of life e.g. quality, yield and time to market that can qualifyfor organic certification e.g. OMRI.

The economic impact for the use of this invention is documented in termsof feature/benefits. Solutions to the recurring problem of plantpathogens have been explored for decades. As particular crops becomemore abundant, and the area of land allocated for agriculture expands,or as greenhouse needs expand, there is an inherent need to employ moreefficient and effective agronomic practices, preferably thosebeneficials provided by Mother Nature herself and that occur naturallyin our global environment.

As a result of increasing demand for crop production, farmers must oftencompromise their cultural practices by planting crops on sub-optimalland, or by increasing the frequency at which crops are planted in aspecific location. In doing so, crop nutrients are depleted, a microbialshift occurs and specific crop pathogens, especially soil-borne orseed-borne pathogens, become more prevalent. Accordingly, it isincreasingly difficult to sustain the health and productivity of crops.It has been well documented that the fungal species C. rosea occurs mostabundantly in virgin soils, wherever such soils can still be foundaround the world; in depleted or overworked soils C. rosea and otherbeneficial endophytes has been gradually eroded and are nearly orcompletely absent from soils thus reducing soil productivity andhampering farmers from producing productive crops e.g. in some Africansoils. The need for improved soil conditioning and the beneficial chaineffect on global economies could be massive.

California alone has a $1.2 billion a year fruit industry; thestrawberry crop alone is estimated to be worth over $400 millionannually but is under pressure to minimize water/energy inputs andproduce more “organic” crops to minimize pesticide usage.

There is increasing pressure in Europe to use only organically approvedcrop production tools amidst a lack of such tools.

In the instance of field peas as one example of a field “pulse” crop inCanada, there were approximately 1,000,000 hectares/year of field peagrown in western Canada in 1999-2005, estimated at a total farm value ofover $600+ million and other oil seed crops such as Canola when acreageshave increased to over 15 million acres planted annually amidst growinginput problems.

In recent years, the field pea, lentil and other specialty crops ofwestern Canada have been most affected by soil-borne or seed-bornediseases, as well as the Ascochyta complexes of root and foliage. Knownas PRRC (pea root rot complex) diseases, the soil-borne and seed-bornediseases are most commonly caused by the pathogens Fusarium solani f.sp.pisi, Fusarium oxysporum f.sp. pisi, Mycosphaerella pinodes, Rhizoctoniasolani, Sclerotina sclerotiorum, Aphanomyces euteiches, Alternariaalternata and Pythium spp. The incidence of PRRC diseases varies withyear and location, while its severity is largely dependent on climate,crop rotation and cultural practices. The yield losses of pea cropsalone, as a result of PRRC pathogens including foliar infection byAscochyta complex, are frequently devastating with a conservative15%-20% yield reduction translating into an approximate annual loss of$80+ million in areas of western Canada.

The rose and flower industry is estimated to be worth over $5 to $6billion dollars (US) in North America at the grower level. The industrynormally requires some 11-14 weeks turnaround time from cuttings tomarket shipment. The ability to produce a higher quality product with areduction in some 10-20 days to market (as the technology in thisinvention provides) is a significant cost benefit to theflower/horticultural industry.

Greenhouse vegetable crops e.g. tomato, cucumber, peppers, etc in Canadaalone are worth an estimated $1.0 billion and employ over 19,000 people.The US industry is valued at more than 10 times that. Losses toindividual greenhouses e.g. a cucumber grower in a production area of44,500 m², crop productivity is frequently 10-15% lower in areas withdiseased plants than where plants were generally healthy. This losstranslates to more than $375,000 per year.

SUMMARY OF THE INVENTION

The present invention provides for a novel, inventive and applicableprocess and method for producing endophytes, specifically a fungusClonostachys rosea as effective plant inoculants that provide for thepromotion of plant vigor, health, growth and yield. Specifically theinvention provides for:

-   (1) an effective plant inoculant of an endophyte product(s) produced    comprising Clonostachys rosea strain 88-710 with unique inoculant    feature/benefits for the promotion of improved plant vigor, health,    quality, growth and yield-   (2) a process for making the product(s) that provides for various    levels of highly viable/stable spores and mycelial material in a    stable formulation with natural emulsifiers that prevents the    degradation of a living endophyte C. rosea strain 88-710 and allows    rapid site occupation on plants.-   (3) product formulations that allow/enables practical use and    application of the product(s) to seeds, roots, stems, leaves,    flowers, bulbs, etc of plants as a water based sprayable    formulations, as a dusts or spore concentrates for other uses    including application equipment/devices of practical use by growers    e.g. seed treatment or insect vectoring, spray or irrigation    equipment,-   (4) and allows the product(s) (endophyte) when applied to, seeds,    roots, stems, leaves, flowers, wounds or cut surfaces of plants to    act as an inoculant ‘enhanced site occupier and rapid colonizer’    within the tissues of plants-   (5) and endophyte product(s) that provide improved root, leaf, stem    and or vegetative bud (flowers) growth to plants whilst reducing    plant senescence to plants as well as-   (6) for the reduction of environmental or cultural stress to plants    e.g. root loss due to trimming, pruning, cuttings, and seeding,    transplanting, or other stresses with the net result of-   (7) improved crop quality, improved rooting, improved    emergence/germination and faster development to marketability of the    crop and further allows-   (8) the product(s) (as endophytes) can be formulated into    value-added products that provides unique feature/benefits to    provide natural induced plant resistance to fungal diseases such as    powdery mildew, botrytis, root rots, seed seed decay, wilt diseases    and or insects/mites.-   (9) and endophyte product(s) that provide improved root, leaf, stem    and or vegetative bud (flowers) growth to plants as well as-   (10) A strain of Clonostachys rosea i.e. 88-710 with a unique    genetic coding that differentiates the endophyte from other fungal    strains of Clonostachyus rosea or Gliocladium roseum. e.g. a strain    that does not produce the sexual state (teleomorph) as does the    anamorph referred to as Bionectria ochroleuca which is    morphologically indistinguishable from C. rosea.-   (11) An endophyte Clonostachys rosea strain 88-710 acts as an    inoculant to stimulate and have an additive effect with rhizobium on    the production of nitrogen fixing nodules on legumes and growth    enhancement e.g. beans, soybeans, peas, and alfalfa.

The invention relates to a process and method for the production and useof endophytes as plant inoculants products that provide unique inoculantfeature/benefits for the promotion of plant vigor, health, growth andyield comprising the fungal group Clonostachys rosea. I also relates toa process and method for producing economically acceptable quantities ofa fungal conidial preparation of Clonostachys rosea utilizing a discretesolid substrate fermentation system. The invention further relates to anendophyte product(s) produced by such processes and methods. Such aproduct may comprise Clonostachys rosea; strain 88-710 and the processfor making the product(s) provide various levels of highly viable/stablespores and mycelia material in a stable formulation that prevents thedegradation of a living endophyte C. rosea strain. The endophyteproduct(s) comprising Clonostachys rosea, strain 88-710 is unique interms of genetic profile and as an endophyte the strain does not producethe sexual state (teleomorph) as does the anamorph referred to asBionectria ochroleuca which is morphologically indistinguishable from C.rosea. The endophyte product(s) may comprise a solid substrate of, forexample, certain cereals e.g. rye, which contain sufficient naturalemulsifiers in the form of various proteins, lignans, to provide the C.rosea spores/mycelium production with excellent naturaldispersing/wetting/sticker agents that allows for rapid site occupationon/in plants for use in EndoFine® formulations e.g. seed treatments,without having to add other emulsifiers for dispersion and use. Theproduct formulation allows/enables practical use and application of theproduct(s) to roots, stems, leaves, flowers, bulbs, etc of plants as awater-based sprayable formulations or as a dusts for other uses e.g.seed treatment, dusts for insect/mite vectors. The product when appliedto seeds, roots, stems, leaves, flowers, wounds or cut surfaces ofplants enables the endophyte to act as an inoculant ‘enhanced siteoccupier’ within the tissues of plants. The product provides improvedroots, leaf, stem and or vegetative bud (flowers) growth to plants andor enhances/improves the germination and emergence of seeds. The productprovides for reduction of environmental or cultural stress to plantse.g. root loss due to trimming, pruning, cutting or other stresses. Theproduct provides improved crop quality and faster development tomarketability of the crop. The product provides for a reduction in thedependency on chemical pesticides for pest control e.g. control ofBotrytis, Fusarium, Pythium, spp. and the like. The product can be usedfor the production of a variety of greenhouse, horticultural andagronomic field crops. The composition of the invention can be a plantinoculant composition comprising Clonostachys rosea spores, conidia ormycelia in admixture with an agrochemically acceptable diluent orcarrier. The invention also relates to a method of enhancing growth,health vigor or yield of a plant which method comprises applying theplant inoculant composition of the invention to a plant or plant locus.The invention further relates to a method of combating a plant funguswhich method comprises applying an antifungally effective amount of thecomposition of the invention to a harmful fungus by occupying thetargeted infection site or habitat of said fungus or denying the foodsource of said fungus. The endophyte of the invention can comprise theendophyte Clonostachys rosea strain 88-170 which acts as an inoculant tostimulate and has an additive effect with rhizobium on production ofnitrogen fixing nodules on legumes (e.g. beans, soybeans, peas, andalfalfa) and leads to growth enhancement. The endophyte of the inventionwhen comprising Clonostachys rosea strain 88-710 provides wound healingbenefits as an inoculant that can in turn, when formulated in suitableformulations, can provide additional/additive plant protective benefitsagainst a wide range of plant diseases, insects and mites e.g. powderymildew, two-spotted mites, aphids. The endophyte of the invention whencomprising Clonostachys rosea strain 88-710 can be combined with rootinghormones such as indole-3-butyric acid (IBA) to provide inoculant plusrooting benefits to cuttings/transplants of plants. The endophyteClonostachys rosea strain 88-710 can be combined with rooting hormonessuch as indole-3-butyric acid (IBA) to provide inoculant plus rootingbenefits to cuttings/transplants of plants for use with roboticequipment in formulations that do not interrupt robotic sensingequipment by leaving sensor sensitive dust particles on plant materialduring transplanting.

The invention relates to a production and use process whereby endophytesas inoculant products specifically the example of strain 88.710 thatprovides for the enhanced promotion of plant vigor, health, growth andyield. The production method for producing the endophyte, C. roseastrain 88-710, provides for producing economically acceptable quantitiesof a fungal conidial preparation utilizing a discrete solid substratefermentation system; the method also results in effective levels ofinoculum in spore and mycelia in stable and practical/applicable productformulations. Specifically the endophyte e.g. Clonostachys rosea,formerly called Gliocladium roseum, as a specific strain 88-710, whichis genetically and reproductively differentiated from other Clonostachysor Gliocladium strains, is used as an example for providing a high levelof inoculant “site occupation” performance in plants that in turnprovides for improved seed, leaf, stem, root growth and yield byreducing stress factors (environmental as well as cultural) in thegrowth of plants.

The invention also provides the novelty of an endophyte Clonostachysrosea strain 88-170 acting as an inoculant to stimulate and have anadditive effect with rhizobium bacteria on the production of nitrogenfixing nodules on legumes and growth enhancement e.g. beans, soybeans,peas, and alfalfa.

The invention also provides unique use and novelty in that endophytese.g. Clonostachys rosea strain 88-170 can be used/formulated to providewound healing benefits that can in turn, when formulated with suitablesalts, and natural emulsifiers (proteins, lactic acid), can provideadditional/additive plant protective benefits against a wide range ofplant diseases, insects and mites e.g. powdery mildew, two-spottedmites, aphids.

The rapid colonisation and use of formulated Clonostachys rosea strain88-710, e.g. as the brand EndoFine®, provides productionfeature/benefits for plants in terms of, faster emergence/germination,shorter production time, faster harvesting and crop marketability andquality.

The invention applies to various plant species and agronomic crops e.g.roses, cucumbers, tomatoes, peppers, cereal crops, legumes, crucifers,etc. grown under greenhouse or field conditions under a variety ofcultural methods e.g. hydroponics, seeds, cuttings, transplants, fieldcultivation, etc. Specific formulations of Clonostachys rosea provideunique inoculant benefits to plants that reduce plant stress to enhanceover all plant growth, health, yield and quality including thepreventive infection via induced plant resistance to plant/seed diseasesand pests thereby reducing dependency on chemical pesticides for pestcontrol.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sporulation of Clonostachys (EndoFine®) on tissues ofstrawberry plants that were treated by immersion in EndoFine suspension.

FIG. 1 a shows sporulation of Botrytis on tissues of strawberry plantsthat were treated by immersion in EndoFine suspension for 2 minutesplanted in pots and sampled 15 days later Botrytis on tissues ofStrawberry Plants. Plants were treated with 10 g EndoFine®/L.

FIG. 2 shows the effects of Clonostachys rosea and Pseudomonaschlorographis on leaf area of cucumber.

FIG. 3 shows the effects of Clonostachys rosea and Pseudomonaschlorographis on dry mass of roots, shoots and whole plants ofhydroponic cucumbers.

FIGS. 4-8 show the results of greenhouse soybean inoculant trials.

FIG. 9 shows colonisation of rose plant shoots and roots using EndoFinealone and in combination with IBA rooting compound.

FIG. 10 shows root weight gains of rose plant roots and roots usingEndoFine alone and in combination with IBA rooting compound.

DEFINITION OF TERMS

As employed above and throughout this disclosure, the following itemsunless otherwise indicated shall be understood to have the followingmeaning:

Endophyte is defined as “fungi or bacteria that form symptom lessinfections, for part or all of their life cycle within healthy leavesand stems of plants” (Definition by Hawksworth et al, 1995). Thisinvention also indicates that the definition of endophytes such as C.rosea may also include living within plant roots and grow to be part theentire plant rhizosphere and have the ability to reduce or convertminerals into forms more easily absorbed by plants.

Clonostachys rosea formerly Gliocladium roseum is an endophyte fungus asdefined in “Compendium of Soil Fungi” vol 1 Domsch, K. H., Gams, W andAnderson, Traute-Heidi Academic Press 1980 pgs 369-374.

A ‘site occupier endophyte’ is defined as meaning an endophyte(fungus/bacteria) that occupies the apoplast within cells i.e. withincell tissues/membranes.

Inoculant is defined as a beneficial microbe that promotes plant health,size, root growth or increases yield and aids in reducing or eliminatingthe use of chemical fungicides.

A legume nodule e.g. from beans, soybeans, peas, and alfalfa, that occuron the roots of plants that associate with symbiotic bacteria undernitrogen limiting conditions.

A symbiotic relationship is defined as a close, prolonged associationbetween two or more different organisms of different species that may,but does not necessarily, benefit each member or a relationship ofmutual benefit or dependence.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the invention is detailed in summary of theinvention (above) and in the claims.

The present invention provides a method and process for the productionand use of:

-   (1) an effective plant inoculant of an endophyte product(s) produced    comprising Clonostachys rosea strain 88-710 with unique inoculant    feature/benefits for the promotion of improved plant vigor, health,    quality, growth and yield-   (2) a process for making the product(s) that provides for various    levels of highly viable/stable spores and mycelia material in stable    but naturally emulsified formulations that prevents the degradation    of a living endophyte C. rosea strain 88-710 while providing    excellent dispersal characteristics.-   (3) product formulations that allow/enables practical use and    application of the product(s) to seeds, roots, stems, leaves,    flowers, bulbs, etc of plants as a water based sprayable    formulations, as a dusts or spore concentrates for other uses    including application equipment/devices of practical use by growers    e.g. seed treatment or insect vectoring, spray or irrigation    equipment,-   (4) and allows the product(s) (endophyte) when applied to, seeds,    roots, stems, leaves, flowers, wounds or cut surfaces of plants to    act as an inoculant ‘enhanced site occupier and rapid colonizer’    within the tissues of plants-   (5) and endophyte product(s) that provide improved root, leaf, stem    and or vegetative bud (flowers) growth whilst reducing plant    senescence to plants as well as-   (6) for the reduction of environmental or cultural stress to plants    e.g. root loss due to trimming, pruning, cuttings, and seeding,    transplanting, or other stresses with the net result of-   (7) improved crop quality, improved rooting, improved    emergence/germination and faster development to marketability of the    crop and further allows-   (8) the product(s) (as endophytes) can be formulated into    value-added products that provides unique feature/benefits to    provide natural induced plant resistance to fungal diseases such as    powdery mildew, botrytis, root rots, seed seed decay, wilt diseases    and or insects/mites.-   (9) and endophyte product(s) that provide improved root, leaf, stem    and or vegetative bud (flowers) growth to plants as well as-   (10) A strain of Clonostachys rosea i.e. 88-710 with a unique    genetic coding that differentiates the endophyte from other fungal    strains of Clonostachyus rosea or Gliocladium roseum. e.g. a strain    that does not produce the sexual state (teleomorph) as does the    anamorph referred to as Bionectria ochroleuca which is    morphologically indistinguishable from C. rosea.-   (11) An endophyte Clonostachys rosea strain 88-710 acts as an    inoculant to stimulate and have an additive effect with rhizobium on    the production of nitrogen fixing nodules on legumes and growth    enhancement e.g. beans, soybeans, peas, and alfalfa.

Examples 1 to 3 (below) summarize the method for producing and preparinga stable effective (colonising) endophyte inoculant product containingClonostachys rosea strain 88-710.

Example 4 details the means by which the endophyte C. rosea when appliedas an inoculant allows the inoculant to act as an ‘enhanced siteoccupier’ within the tissues of plants. This action in turn provides forimproved plant growth of roots, leaf, stem, and/or vegetative bud growth(flowers or fruit) as illustrated in examples 5 and 6. In Tables 1 and4, and 5 trials (commercial greenhouse operations involving over 150,000potted miniature rose plants) demonstrated the consistent activity of C.rosea to improve quality of production in terms of more flower buds,less senescent leaves and shorter growth period for marketability.

Examples 7 to 11, illustrate the reduction of environmental or culturalstress to plants e.g. root loss due to trimming, pruning, cuttings,seeding, transplanting, or other stresses with the net result of (7)improved crop quality and faster development to marketability of thecrop. Pruning of rose plants to stimulate bud development usuallyresults in a 40-50% loss of net root mass; the resulting stress to theplants causes the plants to produce sugars to compensate for thetrimming stress (note: similar effects occur to plants from variousenvironmental or cultural stresses). This in turn makes rootssusceptible to root diseases e.g. fusarium, pythium. C. rosea acts as aninoculant “site occupier” to prevent the sugars from being consumed byharmful plant pathogens and in this way allows the sugar reserves of theplant to maintain existing root mass and promote faster vegetative stemand leaf growth . . . hence faster yield, marketability and higherquality plants.

Preparation of a Stable Effective Endophyte Inoculant Product ContainingClonostachys rosea

EXAMPLE 1

The unique method of preparation and know-how linked to the performanceclaims in this invention involves identifying performance standards thatproduce: ** ** (also note references: Schroeder's, H. J. Samuels, G. J.,Siefert, K. A. & Gams, W. (1999) Classification of the myoparasiteGiocladium roseum in Clonostachys as C. rosea, its relationship toBionectria ochroleuca and notes on other Gliocaldium like fungi.Mycologia 91, 365-383. Domsch, K. H., Gams, W., Anderson, Traute-Heide(1980) Compendium of Soil Fungi Volume 1, Academic Press, A Subsidiaryof Harcourt Brace Jovanovich, Publishers)

-   -   (1) A percent acceptable recovery method of a minimum of 60%        conidia/viable spores from a solid matrix.    -   (2) An analytical range: of 0.1 to 5×10 to the 9^(th) (or        higher) CFU for pure inoculum powder (concentrate). Preferred        concentrations range from 10⁴ to 10¹² CFU/gram, especially from        10⁶ to 10¹⁰ CFU/gram, particularly from 10⁷ to 10¹⁰ CFU/gram,        and more especially from 10⁷ to 10⁹ CFU/gram.    -   (3) A method to measure a minimum limit of quantification of 2        to 3.7×10 to the 8^(th) CFU/gram (or higher) for finished        end-use formulation. Finished end-use formulations usually are        diluted by admixture with an agrochemically acceptable diluent.        Dilution can be anything from 2 to ten thousandfold (usually)        though dilutions of between 10 and a thousandfold are more        preferred and dilutions of 15 to 100 times are especially        preferred in most situations.    -   (4) A limited of detection for viable spores of 0.1 to 5×10 to        the 9^(th) and higher    -   (5) % CV repeatability: 85% (within lab) or higher    -   (6) % CV reproducibility (between lab) 85% or higher

Production of Viable Conidia by Solid Substrate Fermentation

The preparation of a stable and effective endophyte inoculant containingof Clonostachys rosea involves cultivation of Clonostachys rosea cellson a solid substrate. Preparation of Fresh Vegetative or ConidialCultures of C. Rosea (Inoculum), is Carried Out by incubating a pureculture on a suitable nutrient media such as Potato Dextrose Agar orMalt Extract Agar (PDA source is from Adjuvants Plus Inc., Malt Extractcan be obtained from one or more commercial suppliers), and allowing thecultures to incubate/sporulate for about 7-20 days depending ontemperature and humidity. Alternatively, cultures may be transferred toone or more liquid or solid substrate media such as cereal grain,nutrient enriched vermiculite or other solid matrix, and allowed tocolonize said substrate in order to facilitate improved dispersionduring inoculation.

A particulate material (or a mixture thereof) capable of holdingsufficient moisture and nutrients required to sustain the growth andpromote sporulation of C. rosea cultures is introduced into a rotaryblender capable of achieving and maintaining temperatures and pressuresrequired for sterilizing material. A typical mixture contains a cerealgrain or other solid substrate, such as rye, millet, or vermiculite,chalk or gypsum and sufficient water to produce a finished productcontaining approximately 45-55% moisture and a pH between 6.6 and 7.6. Aunique feature in this invention) of the solid substrate, is thatcertain cereals e.g. rye, contain sufficient natural emulsifiers in theform of various proteins, lignans, to provide the C. rosea spores withexcellent natural dispersing/wetting/sticker agents for use in EndoFine®formulations e.g. seed treatments.

The nutrient mixture is blended and sterilized by heating toapproximately 125° C. and maintained at this temperature for 20-45minutes as necessary to achieve adequate sterilization. The sterilizedmixture is permitted to cool to a temperature below 50° C. and C. roseainoculum as described above, is introduced aseptically into the blendingvessel. Once introduced, the inoculum is thoroughly mixed and dispersedthroughout the sterile substrate material, connected to a bag fillingsystem by means of a sterile connector and filled into sterilepolyethylene bags containing microporus breathing strip. The filled bagsare immediately sealed in a clean room, and transferred to a suitablegrowing environment for incubation and allowed to grow to maturity.

Alternatively, the particulate material can be premixed, introduced intobags, trays or other suitable containers and sterilized insitu. Thesterile particulate substrate can then be admixed with a suitable C.rosea inoculum and allowed to grow to maturity. Prepared and inoculatedsubstrate, in bags is incubated at room temperature 18-24° C. until thesubstrate is densely colonized with mycelium of C. rosea (about 6 days);care is taken to allow for air exchange, to eliminate excess water frombags and allow for even growth of mycelium over a period of 7-28 daysdepending on conditions. Continued incubation produces spores of thefungus with 6-10 weeks of inoculation depending on growing conditions.The colonized substrate may be periodically shaken to insure theuniformity of growth, promote sporulation and maximize productivity.

After sporulation, the colonized grain is ground and the product issorted via a vibratory sieved into 100, 40 or various mesh sizes;various mesh sizes are used to prepare final formulations depending onuses. The final concentrate of 100, 40 or various mesh sizes of shouldcontain a minimum of 10 to the 10^(th) viable CFU (colony forming units)spores/gram.

A Novel Method for Measuring the Number of Viable Spores per Gram ofProduct EXAMPLE 2

The invention also involved developing a consistent method fordetermining the viable number of spores per gram of powder (CFUspores/g). This was done by place 0.1 g powder in 9.9 ml steriledistilled water in a sterile test tube. (=10 minus 2 dilution). Shakethoroughly, and continuing the dilution series with 1.0 ml in 9.0 mleach time. Zero point one ml (0.1 ml) of each dilution is then ontoPDTSA (in-house source from Adjuvants Plus Inc.) in Petri dishes atleast 3 replicates. The Petri dishes are then incubated for 4-5 daysuntil colonies have developed, and count the colonies. Multiply thecolony count by the dilution factor to give number of viable(colony-producing) spores per gram powder. For example, the firstdilution of 10 to minus 2 becomes 10 to 3 because only 0.1 was used forplating, so in this instance multiply the colony by 1000.

For determining the number of viable spores per gram of grain substrate,1 g of dried grain is sampled randomly from stored inoculum. The grainsare shaken in sterilized distilled water plus surfactant (in sourceAdjuvants Plus Inc.) at 110 rpm for 10 min., filter the spore suspensionthrough 3 layers of cheesecloth, estimate the spore concentration withaid of hemacytometer, and continue the dilution series with 1.0 ml in9.0 ml each time if needed.

Preparation of Stable Useful Product Formulations of C. Rosea EXAMPLE 3

All living fungal/bacterial endophytes are highly susceptible todegradation due to a number of factors e.g. high temperatures (4° C. orhigher), halogens in water e.g. chlorinated water, bacterialcontamination, etc. This invention also involves the preparation of anovel formulation that provides stability for viable spores and theformulation of the products for practical use and application to roots,stems, leaves, flowers, bulbs, etc of plants as a water based sprayableformulations or as a dusts for other uses e.g. seed treatment.

The objective with final formulations is to have stable viable spores inthe finished product at various desired levels i.e. from 0.1 to minimumsof 2×10⁷ spores/g. or higher. The fine powder of concentrated C. rosea(100 mesh) is mixed with an inert carrier (calcium carbonate 15-30% w/w;talc 13-15% w/w and sodium dibutylnaphthalene sulfonate (75-78% w/w and,sodium sulfate 13-20% w/w) for a product example EndoFine® of 14.5% w/wconcentrate powder of C. rosea: 85% w/w carbonate/talc: 0.5% sodiumdibutylnaphthalene sulfonate mix w/w.

Different EndoFine products can also be made from the C. roseaconcentrate e.g. 2.5×10 to the 8^(th) CFU/g or higher for adequatedelivery on seeds and other formulations for a variety of uses.

Basically, the mixing rate is determined by the density of spores of C.rosea contained in powder product. The finished product(s) can be storedin waterproof containers at 4° C. for a period of 1 year or more beforeusage (see example 14). Product can also be moved in and out ofrefrigerated storage and kept at room temperatures for minimum periodswithout affecting degradation or viability of C. rosea in the finishedformulation.

The Endophyte C. rosea as an Inoculant to Act as and Enhanced SiteOccupier within Tissues of Plants and Imparting Plant InoculantResistance to Diseases, Insects and Mites

EXAMPLE 4

The sampling of leaf, stem and root tissue of treated plants fromvarious crops e.g. roses (both field and greenhouse crops see Tables 1to 10, have indicated that product applications of C. rosea (prepared asoutlined above), are able to penetrate and colonize stems, leaves androots. The sampling also indicated that C. rosea was able to growrapidly in naturally senescent or stressed tissues of plants andcontinue to sporulate after treatment. In Tables 1 and 2 (below).Endophytic development of Clonostachys rosea, strain 88-710, wasestablished in treated rose plants of all treatments at all samplingtimes i.e. C. rosea was present inside leaves, stems, and roots. C.rosea was also present in senescent leaves. No other pathogenicorganisms were recovered from leaves and stems incubated in thelaboratory e.g. Botrytis cinerea or Alternaria alternate i.e. the trialswere completed in the absence or near absence of disease to demonstrateinoculant feature/benefits.

Tables 8-10 (below) illustrate the colonization of C. rosea strain88-710 on various varieties of strawberries as an immersion treatment totransplants that were subsequently grown in the greenhouse to maturity.Growth differences as measured in dry mass (all strawberry varieties)treated with EndoFine® was 122% over untreated plants. Table 9illustrates the ability of Clonostachys rosea, strain 88-710 to impartnatural plant resistance to diseases to treated plants Vs. untreatedplants e.g. botrytis in strawberries after a transplant dip treatment(also see example 18, Table 18 re protection against powdery mildews,insects and mites).

The Endophyte Product of C. rosea Providing Improved Seed Growth,Germination, Root, Leaf, Stem and or Vegetative Bud (Flower) Growth toPlants and Improved Quality

The following examples (Tables 1 to 13, and 19) demonstrate the use ofClonostachys rosea, strain 88-710 for providing improved seedgermination, seed, root, stem and or vegetative growth benefits toplants and improved crop quality/harvest and yield.

EXAMPLE 5

TABLE 1 Trials on rose varieties**; number of flowers, flower buds,percent of dead or senescent leaves and quality index in miniature rosestreated with Clonostachys rosea at 80 days after planting (treatments atrates of 15 g/Litre of finished product (2 × 10⁷ spores/g of freshcuttings, planted cuttings, first trimming, second trimming orcombinations thereof). The treatment in the table below has beenreplicated 8 times for various rose varieties in commercial greenhousetrials involving over 50,000 planting pots (4 inches in diameter eachcontaining 4 plants). Flower buds Open flowers Senescent/dead Treatmenttiming (number/pot) (number/plot) leaves (%) Quality index 1. Nottreated 8.3^(c) 1.3^(c2) 10.3² 5 2. Fresh cuttings (FC) 11.3^(bc)2.3^(bc) 4.7^(bc) 7 3. Planted cuttings (PC) 9.0^(c) 1.3^(c) 8.0^(ab) 74. First trimming (FT) 10.7^(c) 3.3^(ab) 4.3^(c) 7 5. Second trimming(ST) 15.0^(ab) 1.7^(c) 4.3^(c) 8 6. FC + FT 10.0^(c) 1.0^(c) 5.0^(bc) 87. FC + ST 15.7^(a) 4.0^(ab) 3.3^(c) 8 8. FC + FT + ST 14.0^(ab) 6.3^(a)1.3^(d) 10 9. PC + FT 15.3^(ab) 3.0^(bc) 4.3^(c) 9 10. PC + ST 15.7^(a)4.7^(ab) 4.0^(c) 9 11. PC + FT + ST 17.7^(a) 5.3^(a) 1.3^(d) 10 Valuesfollowed by the same letter are not significantly different (PLSD, p >0.05) ** roses (Parade series, Padio Hit, Town and Country, Courtyard,Vigorosa, Flower Circus. Climbing Max, various cultivars from Denmark,Germany)

EXAMPLE 6

In Tables 2 and 3 (below), experiments were initiated when roots ofhydroponic pepper plants had grown 5-8 cm upstream and downstream fromthe rockwool plugs in the baskets, and before roots of adjacent plantsmade contact. For treatments, each beneficial microbe was added to thenutrient solution in the reservoir of each of four replicate hydroponicunits. Final density of the bacteria was 10⁷ cells mL⁻¹ of the totalnutrient solution in the unit. C. rosea density was 10⁶ spores mL⁻¹ oftotal nutrient solution.

TABLE 2 Effects of beneficial microbes on growth of pepper plants insmall-scale hydroponic trough systems with treatments of _(C. rosea)Root Root Root Leaf Plant Shoot volume fresh dry area height freshTreatment (mL) mass (g) mass (g) (cm²) (cm) mass (g) Control 10.5 b 4.7b 0.27 b 1410 b 33.7 b 57 b Ps. chlororaphis Tx-1 14.9 a 7.1 a 0.44 a1667 a 40.2 a 66 ab Ps. fluorescens 63-28 16.4 a 7.5 a 0.45 a 1515 ab41.7 a 69 a C. rosea 88-710 17.3 a 8.2 a 0.51 a 1743 a 42.0 a 75 a Meansin a column followed by the same letter are not significantly different(protected LSD, P < 0.05).

TABLE 3 Effects of beneficial microbes on growth of pepper plants insmall-scale hydroponic trough systems. Root Root Root Leaf Plant Shootvolume fresh dry area height fresh Treatment (mL) mass (g) mass (g)(cm²) (cm) mass (g) Control 25 a 20.7 b 1.35 a 2055 c 51 b 136 b Ps.chlororaphis Tx-1 27 a 21.8 b 1.30 a 2419 b 55 ab 138 b Ps. fluorescens63-28 28 a 23.9 ab 1.37 a 2666 a 60 a 146 a C. rosea 88-710 29 a 24.6 a1.41 a 2714 a 61 a 144 ab Means in a column followed by the same letterare not significantly different (protected LSD, P < 0.05).The Endophyte Product of C. rosea Providing for Improved Crop Qualityand Faster Development to Marketability.

EXAMPLE 7

TABLE 4 Commercial greenhouse trials on varies rose varieties; Effectsof Clonostachys rosea treatments on numbers of flower buds and openflowers, percent senescent/dead foliage, and plant appearance quality inminiature roses at 80 days after cuttings were planted. Treatments wereat rates of 10 g/Litre of finished product (2 × 10⁷ spores/g) of freshcuttings, planted cuttings, first trimming, second trimming on 50,000 (4inch) rose pots each containing 4 plants. Flower buds Open flowersSenescent/dead Quality index Treatment timing (no. per pot) (no. perpot) leaves (%) (1-10)^(a) 1. Not treated 7.6^(c) 1.7^(c) 15.0^(a) 4 2.Fresh cuttings (FC) 10.7^(bc) 3.3^(bc) 5.4^(bc) 7 3. Planted cuttings(PC) 9.3^(bc) 2.7^(c) 6.7^(bc) 7 4. First trimming (FT) 11.0^(bc)4.7^(ab) 4.0^(c) 8 5. Second trimming (ST) 14.0^(ab) 3.3^(bc) 4.0^(c) 86. FC + FT 11.7^(b) 4.3^(abc) 3.7^(c) 8 7. FC + ST 15.0^(a) 5.7^(ab)3.0^(cd) 9 8. FC + FT + ST 15.0^(a) 7.0^(a) 0.7^(d) 10 9. PC + FT15.3^(a) 4.0^(bc) 3.7^(c) 9 10. PC + ST 14.7^(a) 6.0^(ab) 3.0^(c) 9 11.PC + FT + ST 17.0^(a) 6.7^(a) 1.0^(d) 10 ^(a)Considers appearance offoliage and flowers; 1 = very poor, 10 = excellent. All plants in atreatment assessed collectively.

EXAMPLE 8

TABLE 5 Trials on rose varieties; Average Number of the flowers, flowerbuds, percent of dead or senescent leaf and quality index in miniatureroses treated with Clonostachys rosea at 80 days after planting.Treatments with 2 × 10⁷ spores/g at rates of 5 g/Litre at firsttrimming, second trimming; a total of 50,000 (4 inch Size) rose potswere treated each containing 4 rose plants. Flower buds Open flowersSenescent/dead Treatment timing (number/pot) (number/plot) leaves (%)Quality index 1. Not treated 8.0 1.5 12.0 5.0 2. First Trimming (FT)11.0 3.4 3.0 7.5 3. Second Trimming 15.0 2.9 4.0 8.0 (ST)* ready to shipat 65 days

These assessments in Tables 1, 4 and 5 coincided with the general timeat which the grower would ship the plants to buyers. The data on thenumbers of flower buds and open flowers underscored the immensebeneficial impact of Clonostachys rosea in promoting earlier and betterflowering, healthier plants and improved vegetative growth.

The Treated Plants were Shipping-Ready 10-15 Days Before the UntreatedPlants.

EXAMPLE 9

TABLE 6 In replicated treatments on hydroponic cucumbers the followingtreatments were conducted for various inoculants. Experiments*Treatments Concentrations 1 2 3 1 Pseudomonas chlororaphis Tx-1 10⁶cfu/mL X X X (Spotless) 2 Pseudomonas fluorescens 63-28 10⁶ cfu/mL X X X(AtEze) (= Pseudomonas chlororaphis 63-28) 3 Clonostachys rosea(EndoFine ®) 10⁶ cfu/mL X X X 4 Trichoderma harzianum 10⁶ cfu/mL X X X(PlantShield) 5 Bacillus cereus HY06 10⁶ cfu/mL X X 6 Bacillus subtilisGB03 10⁶ cfu/mL X X (Companion) 7 Streptomyces griseoviridis K61 0.05g/L X X (Mycostop) 8 Chitosan 0.05 g/L X 9 Fish hydrolyzate(‘Drammatic’) 15 mL/L X X 10 Controls (not treated) — *Experiments 1, 2,and 3, respectively were conducted in spring/summer, fall, andwinter/spring crop periods. *EndoFine is a registered Trademark ofAdjuvants Plus Inc.

TABLE 7 Effects of four microbial agents/products applied once (1),twice (2), or three times (3) to the root zone of cucumber plants onfruit yield in the spring/summer experiment. Number of No. of fruit/30Yield Agents/products applications plants p-value¹ Change(%)² None(control) — 1069.7bc Pseudomonas chlororaphis 63-28 1 1048.3bc³ 0.579  −2% (AtEze) 2 1136.3ab 0.096 +6.3% 1181.9a 0.007 +10.5%  Bacillussubtilis 1 1054.0bc 0.683 −1.4% 2 1000.0c 0.071 −6.5% 3 1147.7ab 0.051+7.3% C. rosea* strain 88-710 1 1171.0ab 0.013 +9.5% 2 1117.3ab 0.221+4.5% 3 1188.7a 0.005 +11.1%  Trichoderma harzianum 1 1121.3ab 0.096+4.9% 2 1161.7a 0.023 +8.6% 3 1063.0bc 0.867 −0.06%  ¹F-test contrastbetween treatment and control ²Yield change relative to the control³Values followed by the same letter are not significantly different(T-grouping LSD, p < 0.05) *EndoFine ® a registered trademark ofAdjuvants Plus Inc. FIG. 1 shows sporulation of Clonostachys(EndoFine ®) on tissues of strawberry plants that were treated byimmersion in EndoFine suspension for 2 minutes, planted in pots andsampled 15 days later. Plants were treated with 10 g EndoFine ®/L*Sporulation of Clonostachys on the senescent/dead tissues (0-100scales) Clonostachys was well-established in the leaf petioles and rootsof treated plants of all of the tested cultivars (index values 60-100).The leaf laminae were only lightly colonized (10-20), but these weremostly new tissues that had grown AFTER the immersion treatment.

TABLE 8 Sporulation of Clonostachys (EndoFine ®) and Botrytis on tissuesof strawberry plants that were treated by immersion in EndoFinesuspension for 2 minutes, planted in pots and sampled 15 days later.EndoFine Clonostachys index² Botrytis index³ Cultivar treatment¹ LaminaPetiole Root Lamina Petiole Root Annap- − 5 5 0 10 55 0 olis + 10 60 600 20 0 Caven- − 0 0 0 0 60 0 dish + 10 90 75 0 20 0 Jewel − 8 0 0 10 350 + 15 100 70 0 0 0 Kent − 0 15 10 0 30 0 + 10 80 80 5 5 0 Veestar − 5 00 0 25 0 + 20 100 90 0 0 0 − untreated (Control) + treated with EndoFinesuspension ¹10 g EndoFine ®/L ²Sporulation of Clonostachys on thesenescent/dead tissues (0-100 scales) ³Sporulation of Botrytis on thesenescent/dead tissues (0-100 scales)

TABLE 9** Effects of EndoFine ® (C. rosea) on growth components ofstrawberry dipped in EndoFine ® suspension before transplanting andgrown in greenhouse at 77 days. EndoFine Number of Length of Average Drymass (g) Cultivar treatment¹ Runner runner(cm) Root Shoot CrownAnnapolis − 2 60.5 1.03 2.07 1.31 + 5 51.6 1.58 6.03 1.52 Cavendish − // 0.69 2.06 0.79 + / / 1.22 4.19 2.28 Jewel − / / 0.39 0.24 0.77 + 166.8 0.59 4.91 0.71 Veestar − 1 32.7 0.66 2.01 0.63 + 2 50.25 0.93 3.340.76 − untreated (control) + treated with EndoFine ® / not available ¹10g/1 L EndoFine ® **Differences in dry mass (all strawberry varieties)treated with EndoFine ® over untreaed: 122% Strawberry varieties treatedwith 20-8-20 fertilizer every 7 days at 25-100 g/100 L.FIG. 1 a shows sporulation of Botrytis on tissues of strawberry plantsthat were treated by immersion in EndoFine suspension for 2 minutesplanted in pots and sampled 15 days later Botrytis on tissues ofStrawberry Plants. Plants were treated with 10 g EndoFine®/L. AllBotrytis found in the plant tissues would have been from natural sources(chiefly in the field). Index values indicated that leaf petioles ofuntreated plants were moderately infected with Botrytis (25-60), and theleaf laminae lightly infected (5-10) or not infected ((0). Much lessBotrytis was found in petioles of treated plants (0-20) than in petiolesof untreated plants (25-60). No Botrytis was found in roots of untreatedor treated plantsThe Endophyte Product of C. rosea Providing for a Reduction ofEnvironmental or Cultural Stresses to Plants

EXAMPLE 10

In a growth chamber experiment to study the ability of the endophyte C.rosea to withstand environmental and cultural stresses, miniature rosetreated plants taken from treatments (see Table 1) were subjected to 2-3days of limited soil moisture, or given regular watering/plant nutrientsolution. The result was that the untreated plants wilted much moreseverely than treated plants with C. rosea under conditions of limitedroot zone moisture. Plants also took longer to recover in the case ofuntreated plants.

EXAMPLE 11 Stress Due to Trimming

TABLE 10 Percent root mass measurements for several rose varieties(Andrea,) 10 days after trimming. Treatments with 10 g/Litre of finishedproduct containing 2 × 10⁷ spores/g of C. rosea.. The results below werereplicated in approximately 50,000- planted pots (4 inch) in diametereach containing 4 rose plants. % root mass % root mass reductionreduction Quality Treatment timing First trim second trim Index 1. Nottreated and 75% 70% 4.5 trimmed (controls) 2. First Trimming (FT) 25%20% 7.5 Treated 3. Second Trimming (ST) 25% 15% 8.0 Treated; ready toship at 65 days

EXAMPLE 12

TABLE 11 Barley seed treatment field trial for increasingtillering/heads and yield # heads % Difference % difference Treatment:showing per over over (seeds) meter of row untreated Kg Yield untreatedEndoFine 20.3 65% 5302.7 28% 5 g/kg Apron 15.8 28% 4807.9 16% 0.4 mg/kgUntreated 12.3 na 4139.6 naThe Endophyte Product of C. rosea, Strain 88-710 as an Aid to Stimulatethe Production of Nitrogen Fixing Nodules in Legumes for an AdditiveGrowth Effect to Rhizobium Bacteria

Tables 12, 14 and 16 demonstrate the ability of Clonostachys roseastrain 88-710 to act as a seed treatment inoculant to stimulate and havean additive effect with rhizobium on the production of nitrogen fixingnodules on legumes and growth enhancement e.g. beans, soybeans, peas,and alfalfa.

EXAMPLE 13

TABLE 12 Title: Field strip trial seed treatment Field Peas, EndoFineseed treatment 2005 Lethbridge Alberta Variety: Eclipse All seed treatedwith Phiom Bios Tag Team (Pencillium and Mesorhizobium) 1360 kg/bag ofproduct EndoFine seed treatment: 5 g/10 kg of seed A) Plant Emergence**% Difference Treatment Plants/sq meter over untreated EndoFine 48 108.7%VitaFlo 36  56.5% Untreated 23 an B) Yield Bushels/acre % Difference(27.2 kg/bu) over untreated EndoFine 70 16.6% VitaFlo 66  1.7% Untreated65 an **Plants also shoed increased nodulation by 35% over untreatedFormulations of Clonostachyus rosea, Strain 88-710 Show Excellent ShelfLife and Stability

Table 13 illustrates the shelf life and stability of C. raised strain88-710 and the formulated product Endowing® went stored in arefrigerator at 4° C.

EXAMPLE 14

TABLE 13 Density of viable conidia of Clinostats rose (strain 88-710) inan End fine ® formulation stored in a refrigerator at 4° C.** Months ofDensity of conidia storage (CFU/g) 0 4.9 × 10⁷ 2 4.3 × 10⁷ 3 5.1 × 10⁷ 45.1 × 10⁷ 5 5.0 × 10⁷ 6 5.1 × 10⁷ 7 4.9 × 10⁷ 8 4.1 × 10⁷ 11&12 3.5 ×10⁷ **EndoFine product is over formulated to compensate for any minorloss of spores and has a label guarantee of 2 × 10 to the 7^(th) CFU/gspores.

EXAMPLE 15

TABLE 14 Title: Grower Field Trial nitrogen fixation and growth benefitswith EndoFine Variety: AC Kent Seeding date: Jun. 3, 2006; planted intocorn stubble no fertilizer Seed treatment: only EndoFine; no RhizobiumEndoFine seed treatment: 0.5 g/kg of seed Harvest dates: a) EndoFineOct. 15, 2006 b) untreated Nov. 1, 2006 Multiple randomized samples %Difference over Treatment untreated (A) Average root weight (Fresh) perplant (grams) EndoFine 4.82 17.6% Untreated 4.10 na (B) Average plantheight inches EndoFine 35.52 inches  5.1% Untreated  33.8 inches na (C)Average number of pods per plant EndoFine 21.72 20.1% Untreated 18.08 na(D) Average number of nitrogen fixing nodules per plant EndoFine 24.4829.5% Untreated 18.9 na (E) Average Fresh weight per plant (Grams)EndoFine 57.0 10.5% Untreated 51.6 na (F) Harvest dates (days earlier VsUntreated) EndoFine 15 days (G) Average yield (bu/acre) EndoFine 44.58bushels/acre 15.4% EndoFine treated days soybeans harvested 15 Earlierthan untreated soybeans. Untreated 38.62 bushels/acreClonostachys rosea, Strain 88-710 as an Inoculant for the Uptake ofNitrogen, Phosphate and Potassium Nutrient Solutions.

FIGS. 2 and 3 demonstrate the active uptake of N, P, K nutrientsolutions with C. rosea, strain as measured by leaf net CO₂assimilation, leaf chlorophyll fluorescence, and plant growth inhydroponic cucumbers as measured by leaf area and dry mass differences.

EXAMPLE 16

Plants of cucumber (Cucumis sativus L.) ‘Loustik’ were grown from seedsin rockwool plugs (2.5 cm×2.5 cm×4.0 cm) and transferred to single-planthydroponic units when 8-10 cm tall. In each unit, a plant was positionedwith the plug in a hole in the centre of the lid of a 1.9 L whiteplastic container that was filled with nutrient solution (N:P:K,20:8:20; pH 6.0; electrical conductivity 2.5 mS·cm⁻¹). The container wasinserted into a black plastic pot, and the lid and plug were coveredwith black-on-white plastic sheeting, black side downwards. Compressedair with flow regulated by an aquarium air valve was bubbledcontinuously into the solution in each container to maintain thedissolved oxygen level near 8.6 mg/L. The plants were kept in a researchgreenhouse with air temperature near 25° C. during daytime (0800-1600hours) and 22° C. at night. Temperature of the nutrient solution rangedfrom 22.0-23.6° C.

Inoculum of C. rosea and Ps. chlororaphis was applied separately and incombination to the nutrient solution to determine effects of themicrobes on net CO₂ assimilation, leaf chlorophyll fluorescence, andplant growth. Final concentration of each microbe was 1×10⁵ CFU mL⁻¹nutrient solution. Untreated control plants received no inoculum.

EXAMPLE 17

Title: Greenhouse Soybean Inoculant Trial to show growth promotion withand without a nitrogen fixing rhizobium bacteria as a seed treatment.

TABLE 15 Variety: OAC Kent (Round-Up Ready) EndoFine Seed treatmentrate: 5 g/kg Fertilizer treatment: 20-8-20 applied every 2 days at25-100 g/100 L all treatments Rhizobium seed treatment rate: 0.28 ml per100 g of seed Cell-Tech Nitrogen Inc A) Plant Height cm (multiple reps)(see FIG. 4) % Difference Treatment: Average plant Height over untreatedEndoFine 63.74 19.5% Rhizobium 63.40 18.8% EndoFine + Rhizobium 64.1820.3% Untreated 53.35 na B) Plant Fresh weight leaves (multiple reps)(see FIG. 5) Average Fresh weight % Difference Treatment: in grams overuntreated EndoFine 6.23 10.5% Rhizobium 6.66 18.1% EndoFine + Rhizobium7.10 25.9% Untreated 5.64 na C) Plant Fresh weight stems (multiple reps)(see FIG. 6) Average Fresh weight % Difference Treatment: in grams overuntreated EndoFine 3.776 20.6% Rhizobium 3.912 25.0% EndoFine +Rhizobium 4.184 33.7% Untreated 3.13 na D) Plant Leaf Area(multiplereps) (see FIG. 7) Average Leaf % Difference Treatment: Area per plantsq cm over untreated EndoFine 452.22 16.0% Rhizobium 467.26 19.9%EndoFine + Rhizobium 502.54 28.9% Untreated 389.74 na E) Plant dryweight leaves + stems(multiple reps) (see FIG. 8) Average Plant dryweight % Difference Treatment: grams Leaf + stem over untreated EndoFine1.787 19.8% Rhizobium 1.948 30.6% EndoFine + Rhizobium 2.026 35.8%Untreated 1.492 naThe Endophyte Clonostachys rosea, Strain 88-710 Formulations thatProvide Additional/Additive Plant Protection Benefits Against PlantDiseases, Insects and Mites

EXAMPLE 18

Tables 16. Examples A, B, C, D and F demonstrate the performance of C.rosea strain 88-710 on protection against powdery mildew(s), two spottedmites, and red aphids re use cucumber, roses in grower greenhousetrials.

TABLE 16 Clonostachys rosea strain 88-710 wound healing benefits asinoculant formulations for added value plant protective benefits againstdisease, insect and mites. A) Cucumber Grower Trials (Greenhouse) forpowdery mildew Erysiphe cichoracearum; Erysiphe spp,. Sphaerothecafuliginea.) 5 replicates ADJ 702 applied at 16 g/L once to foliageAverage % reduction of mycelium/spores Days after treatment Treatment: 57 10 ADJ 702 99.7 99.7 99.0 (formulation of C. rosea) Untreated 0 0 0Chemical Fungicide 90 70 50 Programs (thiophanate methyl, myclobutanil,pyraclostrobin + boscalid)* *powdery mildew complex believed to beresistant to fungicide sprays; some injury to foliage **ADJ 702 safetyto cucumber foliage was excellent B) Trials on Roses (10 replicates ingreenhouse grower) powdery mildew Sphaerotheca pannosa var. rosea ADJ700 applied at 16 g/L to foliage Average % reduction of mycelium/sporesDays after treatment Treatment: 5 7 10 ADJ 702 98.0 99.5 99.0(formulation of C. rosea) Untreated 0 0 0 Chemical Fungicide 80 60 40Programs (thiophanate Methyl, sulfur, dodomorph)* *powdery mildewfungicides believed to be resistant to powdery mildew and some foliageinjury to rose plants/tender leaves from chemical treatments **safety torose foliage (all stages excellent) C) Trails on roses for red aphidreductions (5 grower greenhouse trails) Macrosyshum spp., Aphis spp.Myzaphis spp. ADJ 702 32 g/L treatment twice (within 2 days) to foliageAverage % reduction aphids/eggs) Days after treatment Treatment: 2 5 1015 ADJ 702 (two applications) 90 95 98 98 (formulation of C. rosea)Untreated 0 0 0 Chemical insecticide 50 40 20 Programs* *aphids believedto be resistant and some foliage injury to rose plants **plant safetyfor ADJ 702 applications excellent D) Residual effect on protection frompowdery mildews, cucumbers greenhouse trials Average Disease (mycelium/spores) % control Days after treatment Treatment: 5 7 10 ADJ 702 98.099.5 99.0 (formulation of C. rosea) 16 g/L spray Untreated 0 0 0Chemical Fungicide 80 60 40 Programs (thiophanate Methyl, sulfur,dodomorph) E) Grower trials on roses (greenhouse) for mite protection(two spotted mite, Tetranychus urticae) ADJ 702 16 g/L treatment twice(within 2 days) to foliage Average % reduction adult mites,crawlers/eggs) Days after treatment Treatment: 2 5 10 15 ADJ 702 (twoapplications) 95 98 99 99 (formulation of C. rosea) Untreated 0 0 0Chemical insecticide 50 70 80 Programs* *mites believed to be resistantand some foliage injury to rose plants from chemical treatments **plantsafety for ADJ 702 applications excellent F) Grower trials on cucumbers(greenhouse) for mite protection (two spotted mite, Tetranychus urticae)ADJ 702 16 g/L treatment twice (within 2 days) sprays to foliage Average% reduction adult mites, crawlers/eggs) Days after treatment Treatment:2 5 10 15 ADJ 702 (two applications) 98 98 99 99 (formulation of C.rosea) Untreated 0 0 0 Chemical insecticide 70 70 80 Programs* *mitesbelieved to be resistant treatments **plant safety for ADJ 702applications excellentThe Endophyte Clonostachys rosea, Strain 88-710 can be Combined withRooting Hormones to Provide Added Value Rooting Benefits toCuttings/Transplants/Grafting of Plant Tissue.

EXAMPLE 19

FIGS. 9 and 10 demonstrate that Clonostachys rosea, strain 88-710 can beused in combination with rooting hormone products such as IBA(indole-3-butyric acid) for the benefit of rooting and inoculantbenefits i.e. C. rosea colonization and plant growth. FIG. 9 showscolonisation of rose plant shoots and roots using EndoFine alone and incombination with IBA rooting compound. FIG. 10 shows Root weight Gainsof rose plant roots and roots using EndoFine alone and in combinationwith IBA rooting compound.

EXAMPLE 20

ENDOFINE concentrate as a dry seed treatment for enhancement of seedgermination and plant growth (growth room trials).

TABLE 17 Seed treatment with Endofine ™ on emergence and roots weight ofsoybean Weight of Roots (g) % Average diff. % Diff. Over untreatedTreatment Emergence (%) Over untreated 10 days 20 days 30 days 10 20 30days CK1* (untreated) 55 na 3.52 4.23 5.76 na E-D* (EndoFine) 95 72.74.28 4.60 6.96 21.5 8.7 20.8 Density of CFU of C. rosea recovered fromthe rhizosphere of the seed treated soybean in Mix-potre soil in growthroom CFU/1000 Colonisation days after Plant part Colonisation of seed 1020 30 E-D-P (EndoFine primary roots) 4.67 × 10 to the fourth 88 12 160E-D-S (EndoFine secondary roots) spores per seed 70.5 1 10 E-D-F(EndoFine fibrous roots) 0 2 25 Untreated 0 0 0 0 Soybean: Variety ACKent; 4 replicates × 7 treatments × 4 samplings = 112 plants Microbialagent and application E-D = Endofine ® dry powder 3.7 × 10⁸ CFU/g @ 5g/Kg of seed as a coating (4.7 × 10 spores/seed) Fertilizer treatment:20-8-20 applied every 2 days at 25-100 g/100 L all treatments

EXAMPLE 21 The Genetic Identification of Clonostachys rosea Strain88-710

Sampling of soils from 18 different locations in Ontario has indicatedthat the 18S rRNA gene sequence of the strain 88-710 with “GeneBank”,has found that the 18S sequence of the 88-710 strain does not haveintron sequence that Bionectria ochroleuca has (See Definitions). Thisindicates that the Clonostachys strain 88-710 does not belong toBionectria ochroleuca. However the strain 88-710 is 100% homologous toC. rosea based on a comparison of internal transcribed spacer (ITS) andthe 28S rRNA gene sequences “GeneBank”. Hence the strain Clonostachysstrain 88-710 does not produce the sexual state (teleomorph) and isgenetically unique.

REFERENCES U.S. PAT. DATE INVENTOR(S) 6,911,338 Jun. 28, 2005 Strobel etal 6,815,591 Nov. 9, 2004 Hignight et al 6,495,113 Dec. 17, 2002 Xue6,475,566 Nov. 5, 2002 Messner et al 6,306,390 Oct. 23, 2001 Narisawa5,723,720 Mar. 3, 1998 Brede at al 5,534,252 Jul. 9, 1996 McAfee et al5,407,826 Apr. 18, 1995 Matsuoka et al 5,068,105 Nov. 26, 1991 Lewis etal 4,550,527 Nov. 5, 1985 Hall et al 4,294,037 Oct. 13, 1881 Mosse et alUS Patent application 20050063955, Mar. 24, 2005; Elmer, Philip AlbertGeorge et al

OTHER REFERENCES

-   1. Domsch, K. H., W. and Anderson. T.-H. 1980 Compendium of Soil    Fungi. Academic Press, London-   2. Pugh, G. J. F., and Dickinson, C. H. 1965. Studies on fungi in    coastal soils, VI, Gliocladium roseum Bainier. Trans. Br. Mycol,    Soc. 48:279-285

1. (canceled)
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 5. (canceled) 6.(canceled)
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 15. (canceled)16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. The method of claim 30 wherein the agrochemicallyacceptable stabilizer carrier comprises an emulsifier.
 22. The method ofclaim 21 wherein the emulsifier is biologically derived.
 23. The methodof claim 22 wherein the emulsifier is cereal grain or an extract orflour thereof.
 24. The method of claim 30 wherein the stabilizedendophyte inoculant composition is in water-based sprayable form. 25.The method of claim 30 wherein the stabilized endophyte inoculantcomposition is in the form of a dust.
 26. The method of claim 30 whereinthe stabilized endophyte inoculant composition comprises the vegetativeor conidial phase of Clonostachys rosea strain 88-710.
 27. The method ofclaim 30 wherein the stabilized endophyte inoculant composition has a pHof 6 to
 8. 28. The method of claim 30 wherein the stabilized endophyteinoculant composition comprises 10⁶ to 10¹² spores/gram of saidcomposition.
 29. The method of claim 30 wherein the stabilized endophyteinoculant composition comprises 10⁷ to 10¹⁰ spores/gram of saidcomposition.
 30. A method of stimulating and providing an additiveeffect with rhizobium on production of nitrogen fixing nodules onlegumes and to enhance plant growth, which method comprises applying astabilized endophyte inoculant composition comprising a vegetative orconidial phase of Clonostachys rosea in admixture with an agrochemicallyacceptable stabilizer carrier to said legumes.
 31. A method of providinginoculant or rooting benefits to a cutting or plant transplant, whichmethod comprises applying a stabilized endophyte inoculant compositioncomprising a vegetative or conidial phase of Clonostachys rosea inadmixture with an agrochemically acceptable stabilizer carrier with arooting hormone to said cutting or plant transplant.
 32. A method ofproviding inoculant or rooting benefits to a cutting or plant transplantwith robotic equipment in formulations that do not interrupt roboticsensing equipment by leaving sensor sensitive dust particles on plantmaterial during transplanting, which method comprises applying astabilized endophyte inoculant composition comprising a vegetative orconidial phase of Clonostachys rosea in admixture with an agrochemicallyacceptable stabilizer carrier with a rooting hormone to said cutting orplant transplant.
 33. The method of claim 31 wherein said rootinghormone is indole-3-butyric acid (IBA).
 34. The method of claim 32wherein said rooting hormone is indole-3-butyric acid (IBA).
 35. Themethod of claim 30 wherein the Clonostachys rosea, strain 88-710 isunique in terms of genetic profile and is an endophyte the strain doesnot produce the sexual state (teleomorph) as does the anamorph referredto as Bionectria ochroleuca which is morphologically indistinguishablefrom Clonostachys rosea.